Peptides comprising aromatic D-amino acids and methods of use

ABSTRACT

Disclosed are D-peptides and libraries of D-peptides comprising aromatic D-amino acids. Also disclosed are methods for identifying small D-peptides comprising aromatic D-amino acids that bind to proteins of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication Serial No. 60/394,176, filed Jul. 3, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] The biological activities of many proteins are modulated bybinding of the proteins to other molecules. For example, lectins are aclass of proteins whose activities are affected by binding tocarbohydrates, including monosaccharides and oligosaccharides. Lectinsare involved in many important functions, including, for example, activetransport and chemotaxis in bacteria, establishing viral infections,mediating leukocyte-endothelial cell recognition, mediating attachmentof bacteria or viruses to other cells, and recognizing normal orpathologic glycoproteins and polysaccharides. Because lectins areinvolved in important biological activities, they are attractive targetsfor drug therapy.

[0004] One approach to identifying a molecule with potential therapeuticvalue is to assess the ability of that molecule to bind to a proteinhaving an important biological activity, because the activity of theprotein may be altered by its binding to a molecule that does notnormally serve as a substrate or ligand for the protein.

[0005] What is needed in the art are new compounds capable of binding toa protein of interest, and methods for identifying compounds having theability to bind to a protein of interest.

BRIEF DESCRIPTION OF THE INVENTION

[0006] In one aspect, the present invention provides a D-peptidecomprising a sequence of from three to seven D-amino acid residues,wherein at least two of the amino acid residues are independentlyselected from the group consisting of D-tryptophan, D-tyrosine andD-phenylalanine.

[0007] In another aspect, the present invention includes a D-peptidecomprising a pentapeptide sequence selected from the group consisting ofXaa₁YYFF, Xaa₁FYFF, Xaa₁YFFF, Xaa₁FFYF, Xaa₁YFFY, Xaa₁YFYF, Xaa₁FFFF,Xaa₁FYYF, FXaa₁FFF, YFXaa₁FF, Xaa₁FWXaa₂Y, Xaa₁FXaa₂WY, Xaa₁Xaa₂FFW,Xaa₁FFFY, FFFFXaa₁, YXaa₁YFF, YXaa₁FFY, Xaa₁FF Xaa₂Xaa₃, Xaa₁WYFF,Xaa₁FXaa₂FF, Xaa₁YXaa₂FF, Xaa₁FFYXaa₂, Xaa₁FFXaa₂F, Xaa₁Xaa₂Xaa₃YY,Xaa₁Xaa₂Xaa₃FF, Xaa₁FYWF, Xaa₁Xaa₂FYY, Xaa₁YYFY, Xaa₁FYXaa₂Y, WXaa₁FFF,Xaa₁FFFXaa₂, Xaa₁YYYY, FXaa₁WFF, WXaa₁FWXaa₂, WFXaa₁FXaa₂, FWXaa₁FF,FXaa₁FFY, Xaa₁Xaa₂WXaa₃Y, FFWXaa₁Y, FXaa₁WXaa₂Xaa₃, YYXaa₁YY, FFFXaa₁F,YFYFXaa₁, YWXaa₁FF, WXaa₁YXaa₂F, WXaa₁YFXaa₂, WXaa₁FFXaa₂, FFFXaa₁W,FWFXaa₁Xaa₂, FYXaa₁YF, FWXaa₁Xaa₂Xaa₃, FXaa₁YYW, FXaa₁YYXaa₂, FWXaa₁WY,FFWYW, FXaa₁Xaa₂FXaa₃, FYWXaa₁Y, FYWXaa₁W, FXaa₁YFXaa₂, FWWYF, FYYYXaa₁,and FFXaa₁WW, wherein Xaa₁, Xaa₂, and Xaa₃ are amino acids of the D- orL-configuration independently selected from the group consisting of D,E, K, R, H, N, Q, S, T, G, A, V, L, I, M, and P.

[0008] In another aspect, the present invention provides a librarycomprising a plurality of D-peptides, wherein each D-peptide comprises asequence of from three to seven D-amino acid residues, wherein thesequences of at least 25% of the D-peptides comprise at least two aminoacid residues independently selected from the group consisting ofD-tryptophan, D-tyrosine, and D-phenylalanine.

[0009] In yet another aspect, the present invention provides a methodfor identifying a D-peptide having the ability to bind to a pre-selectedprotein comprising contacting a library of D-peptides according to thepresent invention with the protein, detecting binding of the protein toa D-peptide to yield a bound D-peptide, and identifying the boundD-peptide.

[0010] In yet another aspect, the present invention provides a methodfor making a D-peptide that binds to a pre-selected protein, comprisingcontacting a library of D-peptides according to the present inventionwith the protein, detecting binding of the protein to a D-peptide toyield a bound D-peptide, identifying the bound D-peptide, andsynthesizing the D-peptide.

[0011] In an important aspect, the invention provides a method forreducing toxicity of a toxin in a mammal exposed to the toxin comprisingdelivering to the mammal a D-peptide of D-amino acids identified asbinding to the toxin in an amount effective to reduce toxicity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

[0013] In one aspect of the invention, the ability of proteins ofinterest to bind to D-peptides comprising D-aromatic acids was evaluatedwith the expectation that D-peptides having therapeutic utility would beidentified. As used herein, a D-peptide is a peptide comprising aminoacids of D-configuration. In addition to D-amino acids, D-peptides mayfurther comprise L-amino acids. By proteins of interest, it is meant anyprotein having or suspected of having biological activity that may bealtered by binding of a molecule to the protein. As discussed above,lectins mediate many important biological functions and therefore, arepotentially useful targets in drug design. Other proteins of interestinclude, without limitation, protein toxins, such as those produced byvarious bacterial pathogens, and antibodies.

[0014] In order to test the ability of D-peptides to bind topre-selected proteins of interest, libraries of pentapeptides enrichedin aromatic D-amino acid residues were synthesized and then tested forthe ability to bind to lectins, various protein toxins, variousantibodies and other proteins. It is envisioned that libraries of shortD-peptides ranging from three to seven amino acid residues in lengthcould also be used to identify a D-peptide that binds to a protein ofinterest.

[0015] For libraries of D-peptides having from three to seven amino acidresidues, a library enriched in D-peptides comprising aromatic D-aminoacids is one in which about 25% or more of sequences in the librarycomprise two or more aromatic D-amino acid residues. Suitably, about 30%or more of the D-peptides comprise two or more aromatic D-amino acidresidues. More suitably still, about 30% or more of the D-peptidescomprise three or more aromatic D-amino acid residues. Still moresuitably, 40% or even as many as 50% or more of the D-peptides compriseat least three or more aromatic D-amino acid residues.

[0016] As described in the Examples below, a pentapeptide libraryenriched in aromatic D-amino acids was constructed in a split synthesismethod using four D-amino acids (alanine, phenylalanine, tyrosine, andtryptophan, or, using the one-letter codes for the amino acids, A, F, Y,and W, respectively) and glycine (G). Glycine is achiral and therefore,does not have D- or L-configurations. As used herein, the A, F, Y and Wamino acids, or other amino acids, are of the D-configuration, unlessotherwise specified. One wishing to create a library enriched inD-peptides comprising aromatic D-amino acids may do so using anysuitable method. About 23% of the pentapeptides in the library made bythe split synthesis method contain two aromatic D-amino acid residues,about 34% contain three aromatic D-amino acid residues, and about 25%contained four aromatic D-amino acid residues.

[0017] For D-peptide sequences in which an amino acid residue may beselected from any one of a number of amino acids, the residue may bedesignated “Xaa₁”. In D-peptides having more than one amino acid residueselected from any one of a number of amino acids, such amino acidresidues will be designated “Xaa₁”, “Xaa₂”, “Xaa₃”, etc.

[0018] Suitably, the D-peptides in a library may be attached to a solidsupport. In the Examples below, a library of pentapeptides enriched inaromatic D-amino acids was synthesized on TentaGel beads, each of whichhas a polystyrene core and, attached to the core, a plurality ofpolyoxyethylene arms, each arm having a primary amine at its free end.D-peptides were synthesized by sequential conjugation of each amino acidresidue added to the D-peptide, using conventional standard D-peptidesynthesis chemistry. The D-peptides thus constructed have free aminotermini. The split synthesis method yields beads each of which comprisesmultiple copies of a single D-peptide sequence. With five amino acids,the number of different pentapeptide sequences in the resulting libraryis 5⁵ or 3125.

[0019] Because the polyoxyethylene arms of the TentaGel beads are watersoluble, the conformations of the D-peptides are determined primarily bythermodynamics and by their primary sequence. As one skilled in the artwill appreciate, the D-peptide may be attached to any suitable support.For example, D-peptides comprising at least one lysine residue at thecarboxy terminus were synthesized and covalently coupled to maleicanhydride-coated 96-well polystyrene plates for use in binding assays.The D-peptides thus coupled to the polystyrene plates have free aminotermini.

[0020] Based on the results obtained in library screenings, summarizedbelow in the Examples, the contribution of the aromatic amino acids F,Y, and W in the D-peptides of the present invention appears to beimportant for binding to proteins. Suitably, a D-peptide according tothe present invention comprises a sequence of from three to sevenD-amino acid residues in length, which sequence comprises at least twoaromatic D-amino acid residues. More suitably, the sequence comprises atleast three or four aromatic D-amino acid residues.

[0021] Although G and A were used as non-aromatic amino acids in theconstruction of the exemplified D-peptide libraries described below, thepresent invention is not restricted to D-peptides or D-peptide librariescomprising G and A residues. As an example, it is specificallyenvisioned that additional D-peptides or D-peptide libraries accordingto the present invention are suitably generated by replacing G and/or Awith any one of the remaining D-amino acids (i.e., D, E, K, R, H, N, Q,C, S, T, V, L, I, M, and P). For example, by replacing G and A withD-serine (S) and D-leucine (L), an additional library of 3125 memberseach could be constructed. It is also envisioned that the G or Aresidues could be replaced with amino acids of the L-configurationproducing libraries of mixed D- and L-configuration peptides.

[0022] It is reasonably expected that G or A could be replaced with“unusual” or “non-natural” amino acids of D- or L-configurations, e.g.,D- or L-α-amino butyric acid, p-chloro-D-phenylalanine,p-chloro-L-phenylalanine, D-(2-naphthyl)alanine, orL-(2-naphthyl)alanine. Such unusual amino acids are commerciallyavailable as derivatives suitable for peptide syntheses. The librarydescribed in the Examples has D-peptides with the amino-terminus as afree amino group. It is envisioned that the free amino group may bederivatized, e.g., acetylated, and the resultant library of peptidestested for binding abilities to any protein of interest. It is furtherenvisioned that a suitable library could be constructed in the samemanner except by eliminating the free amino group at the amino terminiof the D-peptides. This could be accomplished by adding at the last stepof the construction of the library the compounds acetic acid, propionicacid, 3-phenyl-propionic acid, 3-(4-hydroxy-phenyl)-propionic acid or3-indole-propionic acid.

[0023] It is further envisioned that a D-peptide sequence identified asbinding to a protein of interest could be used to design additionallibraries by replacing the non-aromatic residues with other non-aromaticresidues. For example, if a D-peptide having an A residue at aparticular position is identified as binding to a protein, othersublibraries could be readily constructed with permutations at the Aposition. A sublibrary comprising additional D-peptides could beconstructed by replacing A with one of the amino acids not used in theconstruction of the original library. For a D-peptide sequence having Gor A at two or more positions, one could replace the residues at 2positions where a G or A residue is found with different amino acids tocreate a new sublibrary with 196 members. Sublibraries thus createdcould be screened to identify members with different binding specificityor affinity for the protein of interest than the originally identifiedD-peptide.

[0024] An aromatic compound library could also be constructed usingbuilding blocks that are not amino acids. For example, α-hydroxy- orβ-hydroxy-carboxylic acids with aromatic constituents on the α- orβ-carbons could be used and the individual carboxylic acids coupled toeach other via formation of ester bonds. The library could be builtusing the appropriate carboxylic analogues of G, A, F, Y and W (e.g.,glycolic acid, lactic acid, phenyl-lactic acid,3-(4-hydroxyphenyl)-lactic acid or 3-indole-lactic acid) usingcarbodiimide catalyzed couplings, and screened for binding to a proteinof interest as described in the Examples below.

[0025] A suitable library could be built using β-amino acids composed ofthe appropriate analogues of the amino acids G, A, F, Y and W onTentaGel beads in the same manner as done for the D-configurationα-amino acids. Synthesis of D-peptides using β-amino acids analogues wasdescribed in Applella et al., (Nature, 387, 381-384, 1997), which isincorporated by reference herein.

[0026] A pre-selected protein used in screening the D-peptide librariesof the present invention may be any protein of interest, includinglectins, protein toxins, or antibodies, for example. In the Examplesbelow, the jack bean lectin (ConA), the garden pea lectin (PSA), and thelectin designated GSI-B4, as well as two anti-carbohydrate antibodies,were used to screen the D-peptide library for the ability to bindproteins. Competitive binding assays described below in the Examplessuggest that D-peptides may bind to carbohydrate binding sites. However,it should be understood that the present invention is not limited onlyto those D-peptides that bind to carbohydrate binding sites.

[0027] In other Examples, proteins toxins, including botulinum toxins,ricin toxins, cholera toxin, and a component of the anthrax toxin, werescreened for the ability to bind to D-peptides. It is of particularinterest to identify molecules that can interact with toxins such asthese because of the potential for biological warfare using toxins. Foreach toxin tested, D-peptides having the ability to bind to the proteinswere identified.

[0028]Clostridium botulinum produces seven types of botulinumneurotoxins designated BoNT/A-BoNT/G. The toxins inhibit release ofacetylcholine from the pre-synaptic neurons into the neuronal synapse,which may ultimately cause paralysis. Binding of the toxin to cells isrequired for toxicity. Blocking the binding of the botulinum toxins tothe target cells, or blocking the protease activities of theneurotoxins, would prevent or reduce the pathogenic effects of thetoxins.

[0029] In the Examples below, D-peptides that bind to BoNT/A, BoNT/B orBoNT/E were identified. A mixture of three D-peptides having the abilityto bind BoNT/A were administered to mice injected with the BoNT/A toxin.Preliminary data using live mice suggest that the D-peptides reducetoxicity of the BoNT/A toxin in mammals.

[0030] The botulinum toxin binding domain resembles other toxins,including the tetanus neurotoxin (TeNT) (Shapiro et al., J. Biol. Chem.,272, 30380-30386, 1997), diptheria toxin (Choe et al., Nature, 357,216-222, 1992) and Pseudomonas aeriginosa exotoxin A (Allerud et al.,Proc. Natl. Acad. Sci., 83, 1320-1324, 1986). It is therefore expectedthat D-peptides having the ability to bind to TeNT, diptheria toxin, andexotoxin A will be identified using libraries according to the presentinvention, and that such D-peptides may reduce toxicity of the toxins ina mammal.

[0031] Ricin is a plant cytotoxin composed of a cell surface bindingdomain (B) and an enzymatically active A domain with N-glycosidaseactivity (Lord et al., Semin. Cell Biol., 2, 15-22, 1991). The B domainbinds to galactose residues of a cell surface and the A domain cleaves asingle adenine from a conserved sequence of rRNA thus inactivating theribosome and resulting in cell death. (Endo and Tsurugi, J. Biol. Chem.,263, 8735-8739, 1988). The identification of a D-peptide having theability to bind to ricin may reduce binding of the toxin to cells orreduce its activity, thereby reducing toxicity.

[0032] The cholera toxin has one A subunit and five B subunits, and issimilar in overall structure to the E. coli enterotoxin, the Shigelladysenteriae toxin and the Bordetella pertussis toxin. The cholera toxinbinds to cell surface ganglioside GM₁ on the luminal surface ofintestinal epithelial cells, where the A subunit is internalized andmodifies guanine nucleotide-binding proteins involved in regulation ofadenylate cyclase. Blocking the binding of the B subunit to the targetcells will block A subunit internalization and reduce toxicityassociated with the toxin.

[0033] The anthrax toxin has three components: the protective antigen(PA), lethal factor (LF) and edema factor (EF). The PA binds to the hostcell surface receptor, is cleaved by a furin-like protease and thecarboxy-terminal fragment heptamerizes and binds LF or EF (Milne et al.,J. Biol. Chem., 269, 20607-20612, 1994; Elliott et al., Biochemistry,39, 6706-6713, 2000). The EF and LF are translocated to the cytosol ofthe host cell, where EF activates an adenylate cyclase activity and LF,a protease, cleaves members of the mitogen-activated protein kinasefamily. Binding of a D-peptide to a component of the anthrax toxin couldreduce toxicity.

[0034] In other Examples, antibodies were screened for the ability tobind to D-peptides in the D-peptide library. One antibody tested was anantibody which binds to a carbohydrate epitope composed of the H and Leycarbohydrate sequences, which binds to an antigen of endothelial cellsand inhibits activities associated with an angiogenic response(Szekanecz and Koch, Current Opinion _(—) in Rheumatology, 13:202-208,2001). The D-peptide identified as binding to the antibody may be usedto study angiogenesis or to act as an agonist or antagonist ofangiogenesis. A human antibody to an α-Gal epitope involved in theprimate rejection response to transplanted porcine organs (Galili,Biochimie 83:557-563, 2001) was screened to identify D-peptides thatbind to the antibody. Those D-peptides may be useful in blockingrejection mechanisms mediated by the human anti-α-Gal antibodies.

[0035] In other Examples, TNFα and TGFβ1 were screened for their abilityto bind D-peptides, and several D-peptide sequences were identified.TNFα and TGFβ1 are proteins involved in many cell signaling pathways(LaCuca and Gaspari, Dermatologic Clinics 19:617-635, 2001; Taylor,Current Opinion in Rheumatology 13:164-169, 2001; Massague, NatureReview Molecular Cell Biology 1:169-178, 2000; Letterio, Cytokine &Growth Factor Reviews 11:81-87, 2000). The D-peptides identified couldbe used to study signaling pathways or as possible therapeutic agents inpathologies in which TNFα and TGFβ1 are involved as mediators.

[0036] After a D-peptide has been identified as binding to apre-selected protein according to the method of the present invention,one of ordinary skill in the art can readily synthesize the D-peptide insufficient quantity for further evaluation or for use as a therapeutic,which can be used to alter the activity of the pre-selected protein or,in the case of a protein toxin, reduce the toxicity of the toxin.

[0037] For those D-peptides of the present invention intended foradministration to a mammal, (e.g., a mammal exposed to a toxin), theD-peptides are suitably constructed or modified so as to enhancesolubility. In the Examples below, D-peptides administered to mice weredesigned and synthesized to include three D-lysine residues at theC-terminal ends of the D-peptides to enhance solubility. It isenvisioned that from one to four D-lysine residues at the C-terminuswould enhance solubility. It is further envisioned that any amino acidresidue tending to promote solubility could be included at theC-terminus, including R, D and/or E amino acids. It is yet furtherenvisioned that the D-peptides could be derivatized at the C-terminuswith substituents other than amino acids to promote solubility. Suchsubstituents may include a polyoxyethlene polymer or a compoundcontaining multiple hydroxyl groups, such as monosaccharide orpolysaccharide. It is also envisioned that one or more of the D-peptidesmay be chemically coupled to a water soluble compound such as apolysaccharide or protein to promote solubility in water-based solventsor physiologic fluids. It is envisioned that the D-peptides could bephysically incorporated into or chemically coupled to structures such asliposomes in order to promote solubility in water-based physiologicfluids. It is further envisioned that more than one D-peptide could becoupled to a carrier molecule so as to multimerize the resultingconjugated compound for administration to a mammal with the potentialeffect of achieving a functional affinity (avidity) of the D-peptidemultimer. It is yet further envisioned that more than one D-peptideidentified as binding to a protein of interest may be coupled to acarrier compound to potentially achieve functional affinity effects.Additionally, it is envisioned that one or more of the D-peptides may beconjugated to another peptide, protein or carbohydrate sequence (forexample, the sialyl-lactose carbohydrate sequence known to have abinding site on the botulinum neurotoxin) in order to enhance binding ofsuch conjugates to a protein of interest.

[0038] The polypeptide sequence according to the present invention canbe administered in any acceptable manner including orally, parenterally,nasally, by implant, and the like. Oral administration includesadministration in tablets, suspension, implants, solutions, emulsions,capsules, powders, syrups, water composition, and the like. Nasaladministration includes administering the composition of the presentinvention in sprays, solutions, and the like.

[0039] The therapeutic agents useful in the method of the invention canbe administered parenterally by injection or by gradual perfusion overtime. Administration may be intravenously, intra-peritoneally,intramuscularly, subcutaneously, intra-cavity, or transdermally.

[0040] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, intravenousvehicles including fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present, such asantimicrobials, anti-oxidants, chelating agents or inert gases and thelike.

[0041] The actual dosage of a polypeptide sequence of the invention,formulation, or composition will depend on many factors, including thesize and health of an individual. However, the appropriate dosage may bedetermined by one of ordinary skill in the art. The following teachings,which are incorporated by reference, provide guidance: Spilker B., Guideto Clinical Studies and Developing Protocols, Raven Press Books, Ltd.,New York, 1984, pp. 7-13, 54-60; Spilker B., Guide to Clinical Trials,Raven Press, Ltd., New York, 1991, pp. 93-101; Craig C., and R. Stitzel,eds., Modern Pharmacology, d. ed., Little, Brown and Co., Boston, 1986,pp. 127-33; T. Sleight, ed., Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3d ed., Williams andWilkins, Baltimore, 1987, pp. 50-56; R. Tallarida, R. Raffa and P.McGonigle, Principles in General Pharmacology, Springer-Verlag, NewYork, 1988, pp. 18-20. A polypeptide sequence of the invention may beconveniently administered in unit dosage form, and may be prepared byany of the methods well known in the pharmaceutical art, for example, asdescribed in Remington 's Pharmaceutical Sciences (Mack Pub. Co.,Easton, Pa., 1990).

[0042] Physiologically acceptable and pharmaceutically acceptableexcipients and carriers are well known to those of skill in the art. By“physiologically or pharmaceutically acceptable carrier” as used hereinit is meant any substantially non-toxic carrier for administration inwhich a polypeptide sequence of the invention will remain stable andbioavailable when used. For example, the polypeptide sequence of theinvention can be dissolved in a liquid, or dispersed or emulsified in amedium in a conventional manner to form a liquid preparation or mixedwith a semi-solid or solid carrier to form a paste, ointment, cream,lotion or the like.

[0043] Suitable carriers include water, petroleum jelly (VASELINE®),petrolatum, mineral oil, vegetable oil, animal oil, organic andinorganic waxes, such as microcrystalline, paraffin or ozocente wax,natural polymers, such as xanthanes, gelatin, cellulose, or gum arabic,synthetic polymers, alcohols, polyols, water and the like. A watermiscible carrier composition that is substantially miscible in water canbe used. Such water miscible carrier compositions can include those madewith one or more ingredients set forth above but can also includesustained or delayed release carrier, including water containing, waterdispersible or water soluble compositions, such as liposomes,microsponges, microspheres or microcapsules, aqueous base ointments,water-in-oil or oil-in-water emulsions, or gels.

[0044] The carrier can comprise a sustained release or delayed releasecarrier. The carrier may be any material capable of sustained or delayedrelease of the polypeptide sequence. The carrier is capable of releasingthe polypeptide sequence when exposed to the environment of the area ofintended delivery by diffusing or by release dependent on the degree ofloading of the sequence to the carrier in order to obtain release of thepolypeptide of the invention. Nonlimiting examples of such carriersinclude liposomes, microsponges, microspheres, matrices, ormicrocapsules of natural and synthetic polymers and the like. Examplesof suitable carriers for sustained or delayed release in a moistenvironment include gelatin, gum arabic, xanthane polymers; by degree ofloading include lignin polymers and the like; by oily, fatty or waxyenvironment include thermoplastic or flexible thermoset resin orelastomer including thermoplastic resins such as polyvinyl halides,polyvinyl esters, polyvinylidene halides and halogenated polyolefins,elastomers such as brasiliensis, polydienes, and halogenated natural andsynthetic rubbers, and flexible thermoset resins such as polyurethanes,epoxy resins and the like. The sustained or delayed release carrier canbe a liposome, microsponge, microsphere or gel. A pH balanced bufferedaqueous solution for injection can be used. As one of skill in the artwill appreciate, the preferred carrier will vary with the mode ofadministration. The compositions for administration usually contain fromabout 0.0001% to about 90% by weight of the polypeptide sequencecompared to the total weight of the composition.

[0045] The D-peptide libraries may be useful in identifying D-peptidesthat may be used in affinity chromatographic purification of thepre-selected protein of interest. The D-peptides can readily becovalently coupled, using well-known chemistries, to any one of a numberof suitable matrices used in chromatographic separations. The D-peptidematrices could be used to bind to the pre-selected protein from mixturesfollowed by elution and recovery of the protein.

[0046] The following nonlimiting examples are intended to be purelyillustrative.

EXAMPLES

[0047] Peptide Library Design and Synthesis

[0048] A D-peptide library was synthesized by Peptides International,Louisville, Ky. using a TentaGel S resin, NH₂ (“TentaGel beads”). Withthe exception of glycine, which is an achiral molecule, all of the aminoacid residues in the D-peptides are of the D configuration. The TentaGelbeads have a polystyrene core with polyoxyethylene arms attached to thecore; each arm has a primary amine functional group at its terminus. Theresin contains 8.87×10⁵ beads/gram, an average bead diameter of 130microns, 0.2-0.3 meq/gram capacity and 280-330 pmole of primary aminegroups/bead capacity. The amino acids were conjugated to the resin anddeprotected using standard D-peptide synthetic chemistries.

[0049] Amino acids will be designated with the one-letter code. Allamino acids are of the D-configuration unless otherwise noted. Glycinewas attached to the resin to achieve about a 30% substitution of theavailable primary amine groups at the ends of the polyoxyethylene chainsof the Tent-Gel beads. The amine groups to which glycine was not addedwere blocked by acetylation using acetic anhydride. A 30% substitutionyields an average spacing of about 100 to 200 angstroms betweenD-peptides on the bead surface. The spacing was chosen to optimizebinding of a single protein to a single D-peptide sequence, and toreduce the likelihood that steric hindrance will prevent a proteinmolecule from binding to a D-peptide or that a protein molecule willbind to more than one D-peptide.

[0050] Following blocking of the unreacted primary amine groups, theD-peptide library was built by the split synthesis method (Lebl et al.,Biopolymers (Peptide Science), 37, 177-198 (1995). The resin mixture wasdivided equally into five portions and one of G, A, F, Y or W was addedby covalent coupling to one of the five portions of the G-substitutedresin. The beads were then combined, again equally divided into fiveportions, and each portion was used in reactions in which one of G, A,F, Y or W was added in the separate reaction mixtures. The procedure wasrepeated for the five cycles to yield a library of pentapeptidesequences attached to the G residues of the resin. Each bead containedmultiple copies of a single D-peptide sequence. Because five amino acidswere used at each of five amino acid adding steps, the resulting beadlibrary contains 3125 pentapeptide sequences. Following the final aminoacid addition, the resin batches were kept separate, which resulted infive sublibraries of 625 different sequences, designated G, A, F, Y, orW, according to the last amino acid added.

[0051] Screening for Protein Binding to D-peptides and Results ofBinding Assays to the D-peptide Substituted Beads (Peptide-Beads)

[0052] In general, except as otherwise noted, proteins were screened forbinding to the D-peptide beads as follows.

[0053] An aliquot from each sublibrary, each aliquot containingapproximately 1000 beads, was added to a well of a 24-well polystyrenemulti-well plate. From 1.5 to 2 ml Superblock (Pierce Chemical Company,Rockford, Ill.) reagent, 0.1% gelatin (fish skin gelatin, Sigma ChemicalCompany, St. Louis, Mo.), or 1% (w/v) bovine serum albumin (BSA, SigmaChemical Company) in phosphate buffered saline (PBS), pH 7.4, was addedto each well, and the plates were incubated for one to two hours at roomtemperature (RT), with periodic or continuous mixing by gentle rocking.The protein to be tested for binding was diluted in Superblock or 0.1%gelatin-PBS to give a final concentration of about 10⁻⁶ to 10⁻⁸ M. Thediluted protein solution was incubated with the D-peptide-beads for oneto two hours at RT. Following the incubation, the protein solution wasremoved and the beads washed three times with PBS. In the second wash,PBS was left on the beads for about 30 minutes to allow dissociation ofweakly binding protein.

[0054] After washing with PBS, an agent for detecting bound protein wasadded. In some cases, the test protein was labeled with alkalinephosphatase (AP) and no secondary detection agent was required. In othercases, the test protein was labeled with biotin using the biotinylatingreagent NHS-LC-biotin (Pierce Chemical Company) according to thesupplier's instructions. Biotin-labeled protein was detected using APconjugated to neutravidin (Pierce Chemical Company). Another means ofdetecting bound biotinylated proteins used AP-conjugated anti-biotinantibody reagent, which detects bound biotinylated protein on the beads.In other instances, the detection reagent was an AP-labeled antibody tothe protein. The detection agents were generally incubated with thebeads for 30 minutes, after which the beads were washed three times witha Tris-buffered saline solution (pH 7.5), with the second wash beingleft in contact with the beads for 30 minutes. One-step NBT/BCIP(nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphatep-toluidine salt) (Pierce Chemical Co.) was then added and the beadsobserved under a low power microscope until some of the beads had turneda dark purple to purple-black (dark purple-black) color. In the presenceof AP enzyme, the phosphoryl group from BCIP is hydrolyzed and the BCIproduct reacts with NBT which then forms NBT-formazan. The NBT-formazanforms a purple-black precipitate on the beads to which the AP isattached. The beads were then washed with PBS twice, followed by a 1%acetic acid wash, and finally, water. The Fast Red TR/AS-MX substratekit (Pierce Chemical Co.), which yields a bright red precipitate onbeads positive for AP, was used in one experiment. The latterdye-precipitate can be removed by washing the beads with ethanol.

[0055] Dark purple-black beads, or bright red beads (when the Fast Redsubstrate was used), were removed using a small bore pipette andsubjected to amino acid sequence analysis performed at the CoreLaboratories of Louisiana State University Health Sciences Center. Thesequences obtained were essentially unequivocal. Because the fivesublibraries were kept separate, the first residue at the amino-terminuswas known. For all D-peptides in the library, the sixth amino acid is Gbecause G was coupled to the TentaGel beads. For purposes of reportingthe D-peptide sequences, the sixth residue (G) is not reported.

[0056] Binding of Lectins PSA (Pisum sativum, Garden Pea Lectin) andConA (Canavalia ensiformis, Jack Bean Lectin) to D-Peptide-Beads

[0057] The lectins as conjugated with AP (AP-PSA and AP-ConA) werepurchased from EY Laboratories (San Mateo, Calif.). The lectins wereincubated with the F and Y sublibraries by the procedure outlined above.The number of purple-black beads and the number of total beads werecounted in each incubation well, and the percent positive beads wascalculated. The approximate number of positive sequences was calculatedbased on 625 different D-peptide sequences each in the F- andY-sublibraries. TABLE 1 Binding of AP-ConA and AP-PSA Lectins to F and YSublibrary D-peptide Beads Number of positive beads/ Percent 625possible positive sequences AP-ConA F Sublibrary 2.1 13 Y Sublibrary 1.38 AP-PSA F Sublibrary 2.6 16 Y Sublibrary 1.6 10

[0058] The relatively low percentage of positives obtained suggests thatthe binding between the D-peptides of the F and Y sublibraries and thelectins was selective. If proteins bound to the beads only due to thehydrophobicity of the D-peptide sequences, one would have expected toobtain a high percentage of positives. On the other hand, if theproteins had failed to bind to any of the D-peptide sequences, one mightconclude that D-peptides do not fit into the lectin binding sites or toother surface areas of the lectin proteins. Instead, the results showedthat the frequency of binding of the lectins was selective for a smallpercentage of the D-peptide sequences. Control experiments showed thatthe AP enzyme was not responsible for binding to D-peptide sequences.

[0059] The amount of protein binding to a bead was calculated to beabout 5 pmoles protein/bead, based on the following assumptions: (I)AP-PSA and AP-ConA were added in a one ml volume to the beads and at aconcentration of 10⁻⁷ M; (2) the Kd for the D-peptide sequence andlectin complexes is assumed to be about 10⁻⁷ M; (3) one-half of thetotal AP-ConA or AP-PSA protein is bound at equilibrium; and (4) anaverage of about 10 beads out of a 1000 are positive.

[0060] Cross-reactivity of D-peptide-beads for AP-ConA and AP-PSA

[0061] To evaluate the ability of particular D-peptides to bind to bothAP-ConA and AP-PSA, the F- and Y-sublibraries were incubated with eitherAP-ConA or AP-PSA. Positive beads were detected using the Fast RedTR/AS-MX substrate. The positive beads were removed, and the dye washedfrom the beads using ethanol. The original AP-ConA positive beads werethen incubated with AP-PSA, and the original AP-PSA positive beads wereincubated with AP-ConA. Positive beads were then detected using theNBT/BCIP dye reagent and the number of positive beads (purple-blackcolor) was determined. Of 11 beads tested from the Y-sublibrary thatwere initially positive for AP-ConA binding, 3 (27%) were positive forAP-PSA binding. One of 9 (11%) Y-sublibrary initially positive forbinding of AP-PSA was positive for AP-ConA binding. Of 26 beads from theF-sublibrary originally positive for AP-PSA, 8 (31%) were positive forbinding of AP-ConA. Neither of the two beads from the F-sublibrary thatwere positive for AP-ConA binding bound to AP-PSA. Of the total beadstested (48), 12 (25%) were cross-reactive for both lectins. Thus, forlectins that share binding specificities for similar carbohydratestructures, certain D-peptide sequences may exhibit cross-reactivebinding activities. ConA and PSA lectins have specificity for structurescontaining mannose in an α-anomeric glycosidic linkage at thenon-reducing termini of oligosaccharides. It is therefore not surprisingthat certain of the D-peptides to which the lectins bind are the same,and that certain D-peptides may bind to more than one lectin.

[0062] Competitive Binding for Lectins Between D-peptide Beads and theCarbohydrate Ligand

[0063] To test whether the lectins bind to D-peptide sequences throughtheir carbohydrate binding sites, the D-peptide-beads of the F- andY-sublibraries were incubated with AP-ConA in the presence and absenceof 10 mM concentration of α-methyl-mannoside. The beads were thenincubated with NBT/BCIP reagent. In the absence of aα-methyl-mannoside,7.9% and 5.7% of D-peptides of the F- and Y-sublibraries, respectively,bound ConA. When incubated with the D-peptides in the presence ofα-methyl-mannoside, ConA bound to 4.0% and 1.2% of the D-peptides in theF- and Y-sublibraries, respectively. The results suggest thatapproximately half of the positive D-peptide sequences in theF-sublibrary and a fifth of the positive D-peptides in the Y sublibraryD-peptide bind to the same binding site as that to whichα-methyl-mannoside binds.

[0064] In an additional experiment, the F- and Y-sublibrary beads werefirst incubated with AP-ConA in the presence of α-methyl-mannosideyielding 2.1% of the Phe and 1.1% of the Tyr beads as positive. Thosebeads were removed from the incubation wells and the beads furtherincubated with AP-ConA without added α-methyl-mannoside. After thesubstrate NBT/BCIP was added, 3.6% of the Phe and 5.6% of the Tyrsublibraries turned dark purple-black again illustrating that a portionof the D-peptide sequences in each sublibrary were binding to thecarbohydrate binding site of the ConA lectin.

[0065] Binding of Chicken Antibody and a Lectin to D-peptides

[0066] An affinity-purified chicken antibody developed against anantigen comprising an (Gal epitope (Cook et al., J Biosci.& Bioeng., 91,305-310, 2001) and a biotinylated lectin that binds to the same epitope,GS 1, B4 isoform (Murphy and Goldstein, J. Biol. Chem., 252, 4739-4742,1977) were tested for binding to the A- and G-sublibraries. Binding ofchicken antibody to beads was detected using an AP-labeled secondaryantibody to chicken IgY. Binding of the lectin to beads was detectedusing AP-neutravidin. The chicken antibody and lectin were incubatedwith the beads at a concentration of 50 μg/ml, about 0.3 μM and 0.44 μM,respectively. The percentage of D-peptides binding to the antibody orlectin was determined as described above. TABLE 2 Frequencies of Bindingof Chicken Anti-αGal Antibody and the Lectin GS1-B4 to the G- orA-Sublibraries of D-peptide Beads Number of positive beads Percent 625possible positive sequences Chicken anti-αGal A sublibrary 0.07 1 Gsublibrary 0.6 4 GS1-B4 lectin A sublibrary 2.9 18 G sublibrary 3.8 24

[0067] These results show that an antibody to a carbohydrate epitope, aswell as a lectin with a binding site to the same carbohydrate epitope,exhibit specificity in binding to the D-peptide sequences. Furthermore,the results show that a lectin with reactivity to a carbohydrate epitopedifferent from that of ConA and PSA, exhibits binding to D-peptidesequences.

[0068] Binding Specificities of Two Additional Antibodies Reactive withCarbohydrate Epitopes to D-peptide Sequences

[0069] A biotinylated mouse IgM monoclonal antibody to a Ley/Hcarbohydrate epitope (Holloran et al., J. Immunol., 164, 4868-4877,2000) or affinity-purified human anti-αGal antibody, (Fryer et al.,Xenotransplantation, 56:98-109, 1999) were incubated with D-peptidesfrom the A-, G-, F-, Y-, and W-sublibraries. Binding to the D-peptideswas detected using AP-labeled anti-mouse IgM reagent (Sigma) orAP-labeled anti-human Ig reagent (Sigma Chemical Co.). The percentage ofD-peptides binding to the antibodies are shown in Table 3, below. TABLE3 Frequencies of Binding of Two Anti-carbohydrate Antibodies to theD-peptide Beads Number of positive beads/ Sub- Percent 625 possiblelibrary positive sequences Mouse anti- Ley/H A 0 0 G 0.4 3 F 0 0 Y 0.5 3W 0.5 3 AP-labeled anti-mouse IgM reagent A 0 0 G 0.5 3 F 0 0 Y 0 0 W 00 Human anti-αGal A 0.1 1 G 0.2 1 F 0 0 Y 0 0 W 0 0 AP-labeled anti-human IgG reagent A 0 0 G 0 0 F 0 0 Y 0 0 W 0.1 1

[0070] The results show that two additional anti-carbohydrate antibodiesexhibit selective binding to the D-peptide beads. The mouse anti-Ley/Hantibody was reactive with D-peptide sequences of the Y and Wsublibraries. The antibody also bound D-peptides from the G sublibrary,but binding did not exceed background (i.e., AP-labeled anti-mouse IgMreagent bound to the same number of sequences in the presence andabsence of anti-Ley/H antibody). The human anti-αGal antibody appearedto bind to D-peptide sequences of the A and G sublibraries; theAP-labeled anti-human Ig reagent only bound to one sequence of the Wsublibrary. Thus, the D-peptide sequences appear to be specific foranother form of anti-αGal antibody (human) compared to the chickenanti-αGal antibody in the previous example. It was not determinedwhether the human and chicken anti-αGal antibodies bound to the sameD-peptide sequences on the TentaGel beads.

[0071] Preparation of Toxins

[0072] In the Examples that follow, several toxins were screened for theability to bind to D-peptide sequences in the D-peptide bead library.The toxins include the neurotoxin component of the botulinum toxins, thecell binding B subunit of the cholera toxin, the protective antigenportion of the anthrax toxin, and the cell binding component of thericin toxin. These toxins are particularly important because of theirpotential for use in biological warfare agents (J. Am. Med. Assoc., vol.278, no.5, Aug. 6, 1997).

[0073] The neurotoxin components of the A, B and E serotypes of thebotulinum toxins, and the botulinum type B complex form (designatedBoNT/A, BoNT/B, BoNT/E and BotBcomp, respectively) were purified bymethods described by Tse et al. (Eur. J. Biochem, 122, 493-500, 1982)and Moberg and Sugiyama (Appl. Environ. Microbiol., 35, 878-880, 1987).The two forms of the ricin toxin (RCA60 and RCA120) were purchased fromSigma Chemical Co. (St. Louis, Mo.). The cholera toxin B subunit waspurchased from List Biological Laboratories, Campbell, Calif. Theprotective antigen (PA) component of the anthrax toxin was kindlysupplied by the United States Army Medical Research Institute ofInfectious Diseases.

[0074] Frequency of Binding of BoNT/A and BoNT/B to D-peptide Beads

[0075] Biotinylated BoNT/A and BoNT/B neurotoxins were incubated withthe five sublibraries of D-peptide beads, and binding detected using theAP-neutravidin reagent, as described above. The frequencies of strongpositive (purple-black) beads were determined. TABLE 4 Frequencies ofBinding of BoNT/A and BoNT/B Neurotoxins to the D-peptide Beads Numberof positive beads/ Sub- Conc^(#), Percent 625 possible librarv (ug/ml,nM) positive sequences BoNT/A G 100, 667  0.2 1 G 10, 67*  0.3 2 G 10,67*  0 G 10, 67*  0.2 1 G  1, 6.7  0 A 100, 667  0.2 1 A 10, 67*  0 A10, 67*  0 A 10, 67*  0 A  1, 6.7  0 F⁺ 100, 667  0.1 1 Y⁺ 100, 667  0W⁺ 100, 667  0 BoNT/B G 10, 67  2.3 14 G 5, 33  0.5 3 G  1, 6.7  1.0 6 G0.1, 0.67  0 A 10, 67  1.6 10 A 5, 33  0 A  1, 6.7  0.1 1 A 0.1, 0.67  0F 10, 67  0.7 4 F 5, 33  0 F  1, 6.7  0.1 1 F 0.1, 0.67  0 Y 10, 67  0.64 Y 5, 33  0 Y  1, 6.7  0.3 2 Y 0.1, 0.67  0 W 10, 67  1.0 6 W 5, 33  0W  1, 6.7  0

[0076] The results indicate that, as expected, the frequency ofpositives diminishes as the concentration of the toxin incubated withthe beads is decreased below the sensitivity of detection. For example,binding of BoNT/A to D-peptides in the G sublibrary was detectable atBoNT/A concentrations of 667 nM and 67 nM, whereas binding of BoNT/A toD-peptides in the A and F sublibraries was detectable only at a BoNT/Aconcentration of 667 nM. The selectivity of the D-peptides for thetoxins is suggested by the low frequencies of binding. The higherbinding frequencies observed for the BoNT/B toxin may be due todifferential biotinylation of BoNT/B and BoNT/A, to an effect on theBoNT/A activity due to biotinylation, or due to the greater activity ofthe particular purified preparations used in the screening assays.

[0077] Frequencies of Binding of Ricin Toxin (RCA60), Anthrax ProtectiveAntigen (PA) and Cholera Toxin B Subunit (CT) to D-peptide Beads

[0078] The RCA60 form of the ricin toxin, the PA protein and the Bsubunit of the cholera toxin were biotinylated, incubated with theD-peptide library beads, and binding detected using the AP-neutravidinreagent. Numbers of positive beads were counted and the frequenciescalculated. TABLE 5 Frequencies of Ricin (RCA60), Protective Antigen(PA) and Cholera Toxin (CT) Binding to D-peptide Beads Number ofpositive beads/ Sub- Conc^(#), Percent 625 possible library (ug/ml, nM)positive sequences RCA60 G  5, 83 0.5 3 A ″ 0.8 5 F ″ 2.8 18 Y ″ 1.4 9 W″ 2.3 14 PA G 23, 40 0.5 3 A ″ 0.4 3 F ″ 0.4 3 Y ″ 0.2 1 W ″ 0.2 1 CT G0.3 2 A 0 F 0.2 1 Y 0 W 0.4 3

[0079] The results showed selective binding of the D-peptide sequencesto each toxin component tested.

[0080] Additional binding studies were performed with the BoNT/E toxin,with the RCA120 form of ricin, and with the botulinum B complex toxins(BotB complex).

[0081] Sequences of Positive Beads from the Binding Assays with theVarious Proteins

[0082] Positive beads identified in binding assays were selected atrandom and the amino acid sequences determined for the individual beads.The sequences are shown in Table 6. TABLE 6 Sequences of D-peptidesbinding to tested lectins or toxins. Lectin or Toxin Sequences ConAGYYFF; GFYFF BoNT/A GYFFF; GFFYF; GFFYF; GYFFY; GYFYF AFFFF; AFYYF;AFFYF FAFFF YFAFF BoNT/B GFWGY; GFGWY; GAFFW; GFFFY; GFYFF AFYFF; AFFFYFFFFG YAYFF; YAFFY BoNT/E GFFGA; GWYFF BotB complex GFGFF; GYGFF; GFFYG;GFFGF AAGYY; AAAFF RCA60 GFYWF; GGFYY; GYYFY; GFYFF; GYFFY AFYAY; AFYYFWAFFF; WAFFF RCA120 GFFFA AYYYY Cholera FAWFF Toxin WAFWA ProtectiveYGYYA Antigen WFAFG (anthrax toxin) GS1-B4 lectin AFYYF; AFFFA FWAFF;FAFFY Human anti-αGal GAWAY; FFWGY; FAWGA Antibody Anti-Ley/H YYAYYantibody

[0083] Of the total sequences obtained, 90% contain three or fouraromatic D-amino acids. Of those sequences identified from the G and Asublibraries (i.e., D-peptides with G or A residues at theamino-terminus), 89% contained three or four aromatic D-amino acids. Onesequence, GFYFF, was identified as binding to ConA, BoNT/B and RCA60.Another sequence, GYFFY, was identified as binding to BoNT/A and RCA60.A third sequence, AFYYF, was identified as binding to RCA60, BoNT/A andGS1-B4. In two instances, the same sequence was identified as binding toa particular protein: GFFYF for BoNT/A and WAFFF for RCA60.

[0084] Sequences of Positive Beads from Binding Studies using TNFα andTGFβ1

[0085] TNFα and TGFβ1 obtained from commercial suppliers were incubatedwith the D-peptide library beads using the procedures described aboveand binding of the proteins detected using commercially availablemonoclonal and polyclonal antibody antibodies. Positive beads from theTNFα incubations with the F, Y and W sublibraries were removed andsequenced; positive beads from the incubation with TGFβ1 from the Fsublibrary were removed and sequenced. The sequences are listed in Table7. TABLE 7 Sequences of D-peptides Binding TNFα or TGFβ1 TNFα: FFFAF;FFFAF YFAFF; YFAFF; YFAFF; YFAFF; YFYFA; YWAFF WGYAF; WGYFA; WAFFA TGFβ1FFFGW; FWFGA; FYGYF; FWAAA; FAYYW; FGYYG; FWAWY; FFWYW; FAAFG; FYWAY;FYWGW; FAYFG; FYYYA; FWGFF; FFAWW

[0086] The sequence YFAFF from the TNFα screen was found on four of thesix Y sublibrary beads sequenced, and is the same sequence found asbinding BoNT/A. Both beads sequenced from the F-sublibrary of the TNFαbinding study had the identical sequence FFFAF. Two of the 27 totalsequences (7%) contained two aromatic D-amino acids; six (22%) containedthree aromatic D-amino acids; 17 (63%) contained four D-amino acids; andtwo (7%) contained five aromatic D-amino acids.

[0087] Microplate Assay to Determine Protein Binding to D-peptideSequences

[0088] Certain D-peptide sequences identified above as binding toproteins were synthesized with 3 or 4 D-lysine (K) residues at thecarboxyl-terminus in order to increase solubility of the D-peptides inaqueous solutions. The D-lysine-containing D-peptides were covalentlycoupled to maleic anhydride-coated 96-well polystyrene plates (PierceChemical Co.) and the wells were backcoated. Coupling of the D-peptideoccurs predominantly through the D-K amino groups and the majority ofthe D-peptides would then project from the walls of the plate into thesolvent, mimicking the presentation of D-peptides on the TentaGel beads.Proteins were added to the D-peptide-coated wells at variousconcentrations and incubated for at least one hour to allow equilibriumof binding to occur. The wells were washed several times with PBS, andthe relative amounts of protein bound were determined. Usually theproteins were biotinylated and the relative amounts of protein bounddetermined by adding AP-neutravidin and measuring bound AP by incubatingwith p-nitrophenyl-phosphate and measuring p-nitro-phenolcalorimetrically. Maximum binding of proteins was established for thegreater amounts of proteins added to the wells coated with particularD-peptides. Background binding for any protein was determined for wellsnot coated with D-peptide or wells lacking the protein incubation butwith addition of the AP-neutravidin reagent. The dissociationequilibrium constant (Kd) could be estimated from the amount of proteinadded to the D-peptide coated wells that resulted in half maximalbinding.

[0089] The D-peptides used to coat wells, the concentration of toxin atwhich saturation of binding was obtained, and the Kd estimates obtainedfor the proteins bound were as follows. TABLE 8 Determination ofDissociation Constants for D-peptides Binding to Various ToxinsD-peptide Sequences* used for Saturation Coating of Concentra- EstimatedWells Toxin Bound tion, nM Kd, nM GFYFF, AFYAY or RCA60 100  20-25 GFFFYBoNT/A or 3-4  1-2 GFGWY or GAFFW BoNT/A or 3-4 0.5-1  GFFFY BotBcomplex 2.3 0.022

[0090] The results indicate that the D-peptides have high bindingaffinities for the various toxins tested. It is of interest to note thatthe D-peptide sequence GFFFY, was identified as binding to the BoNT/Bneurotoxin and the sequence in this binding assay bound both BoNT/B andBoNT/A neurotoxins as well as the BotB complex. The sequences GFGWY andGAFFW were also identified from D-peptide beads incubated with theBoNT/B neurotoxin and those D-peptides bound both the BoNT/A and BoNT/Bneurotoxins. These results suggest that several of the D-peptidesequences will exhibit cross-reactivites to the structurally similarbotulinum toxins, and that any one of such D-peptides, or mixture ofD-peptides, may be useful for neutralizing the toxic effects of theseveral serotypes of the toxins and the Bot complex form of the toxin.

[0091] Test for Possible Toxicities of D-peptides

[0092] Potential toxicities of D-peptides to be tested for the abilityto neutralize toxins in animals was evaluated by injecting theD-peptides into mice intravenously (iv) or intraperitoneally (ip) andobserving the animals over time for signs of toxicity. TABLE 9D-peptides Used in Toxicity Studies Number Route of Amounts,Concentrations*, Experiment Of mice injection D-peptide(s)^(#) ug mM 1 5iv GFWGY 50 0.025 2 3 ip GFWGY 250 0.125 3 2 ip GFYFF, 640 0.30 AFYAY,WAFFF 4 2 ip GFFYF, 430 0.21 GYFFY 5 3 ip GYFFF, GFFYF, 973 0.46

[0093] In experiments 1 and 2, the mice exhibited no apparent toxicity(e.g., lethargy or ruffled fur) over a five day time period ofobservation. In experiments 3 and 4, the mice appeared to exhibitlethargy for the first 1 to 2 hours following administration of theD-peptides, and then exhibited no apparent signs of toxicity andappeared normal for the remainder of the three-day observation period.In experiment 5, the mice initially exhibited lethargy, ruffled fur andisolationism, then appeared normal on the following day of observation.

[0094] Prolongation of Survival of Mice Injected with Botulinum Toxinplus D-Peptides

[0095] Experiment 1. Two groups of five mice each were injected ip with500×LD₅₀ of BoNT/A neurotoxin alone or the same amount of neurotoxinplus a D-peptide mixture. The D-peptide mixture contained GYFFFKKK (263μg), GFFYFKKK (500 μg), and GYFYFKKK (220 μg). Times to death foranimals in each group were noted. TABLE 10 Survival Times of MiceInjected with Toxin in the Presence or Absence of D-peptides. Times todeath in minutes Animal BoNT/A BoNT/A plus number group D-peptides group1 140 193 2 142 260 3 191 290 4 198 >300 5 >300 >300

[0096] The animals alive at >300 minutes were dead the followingmorning.

[0097] The mean survival times for the five animals given BoNT/A onlywas 194±29 (SEM) minutes (using 300 minutes for the one animal thatsurvived for the initial five hours observation time). The mean survivaltime for the animals given BoNT/A plus the D-peptides was 269±20 minutes(using 300 minutes for the two mice that survived for the initial fivehours of observation time). The p value for the differences in survivaltimes between the two groups by the Students t test was 0.14; the pvalue using the chi square test was 0.11.

[0098] The mean survival times of mice given a large dose of BoNT/A(equivalent to 500× the LD₅₀ the toxin) and treated with D-peptides wasincreased by at least 35%, relative to untreated mice given the samedose of BoNT/A.

[0099] Experiment 2. Experiment 1 was repeated using the same amounts ofBoNT/A neurotoxin (mice injected with 500×LD₅₀ of the toxin). TheD-peptide mixture comprised GYFFFKKK (310 μg), GFFYFKKK (382 μg), andGYFYFKKK (310 μg) with the neurotoxin. Times to death for animals ineach group were noted. TABLE 11 Survival Times of Mice Injected withToxin in the Presence or Absence of D-peptides. Times to death inminutes Animal BoNT/A BoNT/A plus number group D-peptides group 1 117193 2 121 231 3 137 309 4 138 >330 5 165 >330

[0100] Of the two mice that survived greater than the 330 minutes ofinitial observation time, one was dead the next morning and the othermouse survived.

[0101] The mean survival times of the animals given BoNT/A only was135±8.5 (SEM) minutes. The mean survival times of the mice given BoNT/Aplus D-peptides was 278±29 minutes (using 330 minutes as the survivaltimes of the two mice that survived the initial 5.5 hour observationtime). The p value for the difference in survival times was 0.01 usingthe Students t test and 0.009 using the chi square test.

[0102] The mean survival times of the group treated with BoNT/A andD-peptides was double that of the group treated with BoNT/A alone, andthe differences were statistically significant.

I claim:
 1. A D-peptide comprising a sequence of from three to sevenD-amino acid residues, wherein at least two of the amino acid residuesof the sequence are independently selected from the group consisting ofD-tryptophan, D-tyrosine, and D-phenylalanine.
 2. The D-peptide of claim1, wherein the sequence comprises at least three amino acid residuesindependently selected from the group consisting of D-tryptophan,D-tyrosine, and D-phenylalanine.
 3. A D-peptide comprising apentapeptide sequence selected from the group consisting of Xaa₁YYFF,Xaa₁FYFF, Xaa₁YFFF, Xaa₁FFYF, Xaa₁YFFY, Xaa₁YFYF, Xaa₁FFFF, Xaa₁FYYF,FXaa₁FFF, YFXaa₁FF, Xaa₁FWXaa₂Y, Xaa₁FXaa₂WY, Xaa₁Xaa₂FFW, Xaa₁FFFY,FFFFXaa₁, YXaa₁YFF, YXaa₁FFY, Xaa₁FF Xaa₂Xaa₃, Xaa₁WYFF, Xaa₁F Xaa₂FF,Xaa₁Y Xaa₂FF, Xaa₁FFYXaa₂, Xaa₁FFXaa₂F, Xaa₁Xaa₂Xaa₃YY, Xaa₁Xaa₂Xaa₃FF,Xaa₁FYWF, Xaa₁Xaa₂FYY, Xaa₁YYFY, Xaa₁FYXaa₂Y, WXaa₁FFF, Xaa₁FFFXaa₂,Xaa₁YYYY, FXaa₁WFF, WXaa₁FWXaa₂, WFXaa₁FXaa₂, FWXaa₁FF, FXaa₁FFY,Xaa₁Xaa₂WXaa₃Y, FFWXaa₁Y, FXaa₁Wxaa₂Xaa₃, YYXaa₁YY, FFFXaa₁F, YFYFXaa₁,YWXaa₁FF, WXaa₁Yxaa₂F, WXaa₁YFXaa₂, WXaa₁FFXaa₂, FFFXaa₁W, FWFXaa₁Xaa₂,FYXaa₁YF, FWXaa₁Xaa₂Xaa₃, FXaa₁YYW, FXaa₁YYXaa₂, FWXaa₁WY, FFWYW,FXaa₁Xaa₂FXaa₃, FYWXaa₁Y, FYWXaa₁W, FXaa₁YFXaa₂, FWWYF, FYYYXaa₁ andFFXaa₁WW wherein Xaa₁, Xaa₂, and Xaa₃ are amino acids of the D- orL-configuration independently selected from the group consisting of D,E, K, R, H, N, Q, C, S, T, G, A, V, L, I, M, and P.
 4. The D-peptide ofclaim 3, wherein the core pentapeptide is selected from the groupconsisting of GYYFF, GFYFF, GYFFF, GFFYF, GYFFY, GYFYF, AFFFF, AFYYF,AFFYF, FAFFF, YFAFF, GFWGY, GFGWY, GAFFW, GFFFY, AFYFF, AFFFY, FFFFG,YAYFF, YAFFY, GFFGA, GWYFF, GFGFF, GYGFF, GFFYG, GFFGF, AAGYY, AAAFF,GFYWF, GGFYY, GYYFY, AFYAY, WAFFF, GFFFA, AYYYY, FAWFF, WAFWA, YGYYA,WFAFA, AFFFA, FWAFF, FAFFY, GAWAY, FFWGY, FAWGA, YYAYY, FFFAF, YFYFA,YWAFF, FFFGW, FWFGA, FYGYF, FWAAA, FAYYW, FGYYG, FWAWY, FFWYW, FAAFG,FYWAY, FYWGW, FAYFG, FYYYA, FWGFF, and FFAWW.
 5. A library comprising aplurality of D-peptides, wherein each D-peptide comprises from three toseven D-amino acid residues, wherein at least 25% of the D-peptidescomprise at least three amino acid residues independently selected fromthe group consisting of D-tryptophan, D-tyrosine, and D-phenylalanine.6. The library of claim 5, wherein at least 50% of the D-peptidescomprise at least three amino acid residues independently selected fromthe group consisting of D-tryptophan, D-tyrosine, and D-phenylalanine.7. A library according to claim 5, wherein the library comprises atleast five D-peptides.
 8. A library according to claim 5, wherein thelibrary comprises at least ten D-peptides.
 9. A library according toclaim 5, wherein the library comprises at least fifty D-peptides.
 10. Amethod for identifying a D-peptide having the ability to bind to apre-selected protein comprising contacting the protein with a library ofD-peptides according to claim 5, detecting binding of the protein to aD-peptide, and identifying the D-peptide.
 11. A method for making aD-peptide that binds to a pre-selected protein, comprising contactingthe library of claim 5 with the protein, detecting binding of theprotein to a D-peptide, identifying the D-peptide, and synthesizing theD-peptide.
 12. A method for reducing the toxicity of a toxin in a mammalexposed to the toxin comprising delivering to the mammal a D-peptidethat binds to the toxin in an amount effective to reduce toxicity,wherein the D-peptide comprises from three to seven D-amino acidresidues, wherein at least two of the D-amino residues are independentlyselected from the group consisting of D-phenylalanine, D-tryptophan, andD-tyrosine.
 13. The method of claim 12, wherein the D-peptide isidentified according to the method of claim
 10. 14. The method of claim12, wherein the toxin is selected from the group consisting of botulinumtoxins, ricin toxins, cholera toxins, and anthrax toxins or toxinsubcomponents.
 15. The method of claim 12, wherein the toxin is BoNT/Aand the D-peptide comprises a pentapeptide core sequence selected fromthe group consisting of Xaa₁YFFF, Xaa₁FFYF, Xaa₁YFFY, Xaa₁YFYF,Xaa₁FFFF, Xaa₁FYYF, Xaa₁FFYF, FXaa₁FFF, YFXaa₁FF, wherein Xaa₁ is anamino acid of the D- or L-configuration selected from the groupconsisting of D, E, K, R, H, N, Q, C, S, T, G, A, V, L, I, M, and P. 16.The method of claim 15, wherein the toxin is BoNT/A and the D-peptidecomprises a pentapeptide core sequence selected from the groupconsisting of GYFFF, GFFYF, GYFFY, GYFYF, AFFFF, AFYYF, AFFYF, FAFFF,and YFAFF.
 17. The method of claim 12, wherein the toxin is BoNT/B andthe D-peptide comprises a pentapeptide core sequence selected from thegroup consisting of Xaa₁FWXaa₂Y, Xaa₁FXaa₂WY, Xaa₁Xaa₂FFW, Xaa₁FFFY,Xaa₁FYFF, Xaa₁FYFF, Xaa₁FFFY, FFFFXaa₁, YXaa₁YFF, and YXaa₁FFY, whereinXaa₁ and Xaa₂ are amino acids of the D- or L-configuration selected fromthe group consisting of D, E, K, R, H, N, Q, C, S, T, G, A, V, L, I, M.18. The method of claim 17, wherein the D-peptide comprises apentapeptide core sequence selected from the group consisting of GFWGY,GFGWY, GAFFW, GFFFY, GFYFF, AFYFF, AFFFY, FFFFG, YAYFF, and YAFFY. 19.The method of claim 12, wherein the toxin is BoNT/E and the D-peptidecomprises a pentapeptide core sequence selected from the groupconsisting of Xaa₁FF Xaa₂Xaa₃ and Xaa₁WYFF, wherein Xaa₁, Xaa₂, and Xaa₃are amino acids of the D- or L-configuration independently selected fromthe group consisting of D, E, K, R, H, N, Q, S, T, G, A, V, L, I, M, andP.
 20. The method of claim 19, wherein the D-peptide comprises apentapeptide core sequence selected from the group consisting of GFFGAand GWYFF.
 21. The method of claim 12, wherein the toxin is BotB complexand the D-peptide comprises a pentapeptide core sequence selected fromthe group consisting of Xaa₁FXaa₂FF, Xaa₁YXaa₂FF, Xaa₁FFYXaa₂,Xaa₁FFXaa₂F, Xaa₁Xaa₂Xaa₃YY, and Xaa₁Xaa₂Xaa₃FF wherein Xaa₁, Xaa₂, andXaa₃ are amino acids of the D- or L-configuration independently selectedfrom the group consisting of D, E, K, R, H, N, Q, C, S, T, G, A, V, L,I, M, and P.
 22. The method of claim 21, wherein the D-peptide comprisesa pentapeptide core sequence selected from the group consisting ofGFGFF, GYGFF, GFFYG, GFFGF, AAGYY, and AAAFF.
 23. The method of claim12, wherein the toxin is RCA60 and the D-peptide comprises apentapeptide core sequence selected from the group consisting ofXaa₁FYWF, Xaa₁Xaa₂FYY, Xaa₁YYFY, Xaa₁FYFF, Xaa₁YFFY, Xaa₁FYXaa₂Y,Xaa₁FYYF and WXaa₁FFF, wherein Xaa₁ and Xaa₂ are amino acids of the D-or L-configuration independently selected from the group consisting ofD, E, K, R, H, N, Q, C, S, T, G, A, V, L, I, M, and P.
 24. The method ofclaim 23, wherein the D-peptide comprises a pentapeptide core sequenceselected from the group consisting of GFYWF, GGFYY, GYYFY, GYFFY, GYFFY,AFYAY, AFYYF and WAFFF.
 25. The method of claim 12, wherein the toxin isRCA120 and the D-peptide comprises a pentapeptide core sequence selectedfrom the group consisting of Xaa₁FFFXaa₂ and Xaa₁YYYY, wherein Xaa₁ andXaa₂ are amino acids of the D- or L-configuration independently selectedfrom the group consisting of D, E, K, R, H, N, Q, S, T, G, A, V, L, I,M, and P.
 26. The method of claim 25, wherein the D-peptide comprises apentapeptide core sequence selected from the group consisting of GFFFAand AYYYY.
 27. The method of claim 12, wherein the toxin is choleratoxin and the D-peptide comprises a pentapeptide core sequence selectedfrom the group consisting of FXaa₁WFF and WXaa₁FW Xaa₂, wherein Xaa₁ andXaa₂ are amino acids of the D- or L-configuration independently selectedfrom the group consisting of D, E, K, R, H, N, Q, C, S, T, G, A, V, L,I, M and P.
 28. The method of claim 27, wherein the D-peptide comprisesa pentapeptide core sequence selected from the group consisting of FAWFFand WAFWA.
 29. The method of claim 12, wherein the toxin is anthraxprotective antigen and the D-peptide comprises a pentapeptide coresequence selected from the group consisting of YGYYA and WFXaa₁FXaa₂wherein Xaa₁ and Xaa₂ are amino acids of the D- or L-configurationindependently selected from the group consisting of D, E, K, R, H, N, Q,C, S, T, G, A, V, L, I, M and P.
 30. The method of claim 29, wherein theD-peptide comprises a pentapeptide core sequence selected from the groupconsisting of YGYYA and WFAFG.
 31. A method of reducing the ConA lectinbinding to at least one of its receptors comprising delivering to themammal a D-peptide comprising a pentapeptide core selected from thegroup consisting of Xaa₁YYFF and Xaa₁FYFF wherein Xaa₁ is an amino acidof the D- or L-configuration independently selected from the groupconsisting of D, E, K, R, H, N, Q, C, S, T, G, A, V, L, I, M and P. 32.The method of claim 31, wherein each D-peptide comprises a pentapeptidecore sequence selected from a group consisting of GYYFF and GFYFF.
 33. Amethod of reducing binding of GS1-B4 lectin to a GS1-B4 receptorcomprising delivering to the mammal a D-peptide comprising apentapeptide core sequence selected from the group consisting ofXaa₁FYYF, Xaa₁FFFXaa₂, FWXaa₁FF and FXaa₁FFY wherein Xaa₁ and Xaa₂ areamino acids of the D- or L-configuration independently selected from thegroup consisting of D, E, K, R, H, N, Q, C, S, T, G, A, V, L, I, M andP.
 34. The method of claim 33, wherein the D-peptide comprises apentapeptide core sequence selected from a group consisting of AFYYF,AFFFA, FWAFF and FAFFY.
 35. A method of reducing binding of an anti-αGalantibody to an αGal epitope comprising delivering to the mammal aD-peptide comprising a pentapeptide core selected from the groupconsisting of Xaa₁Xaa₂WXaa₃Y, FFWXaa₁Y and FXaa₁WXaa₂Xaa₃ wherein Xaa₁,Xaa₂ and Xaa₃ are amino acids of the D- or L-configuration independentlyselected from the group consisting of D, E, K, R, H, N, Q, C, S, T, G,A, V, L, I, M and P.
 36. The method of claim 35, wherein the D-peptidecomprises a pentapeptide core sequence selected from the group GAWAY,FFWGY and FAWGA.
 37. A method of reducing inhibiting anti-Ley/H antibodybinding to an Ley/H epitope comprising delivering to the mammal aD-peptide comprising a pentapeptide core selected from the groupconsisting of YYXaa₁YY wherein Xaa₁ is independently selected from agroup consisitng of D, E, K, R, H, N, Q, C, S, T, G, A, V, L, I, M andP, the latter amino acids beinfg of D- or L-configuration. 38 The methodof claim 37, wherein the D-peptide comprises a pentapeptide coresequence selected from the group consisting of YYAYY.
 39. A method ofreducing binding of TNFA to a TNFA receptor comprising delivering to themammal a D-peptide comprising a pentapeptide core selected from thegroup consisting of FFFXaa₁F, YFXaa₁FF, YFYFXaa₁, YWXaa₁FF, WXaa₁YXaa₂F,WXaa₁YFXaa₂ and WXaa₁FFXaa₂ wherein Xaa₁ and Xaa₂ are amino acids of theD- or L-configuration independently selected from the group consistingof D, E, K, R, H, N, Q, C, S, T, G, A, V, L, I, M and P.
 40. The methodof claim 39, wherein the D-peptide comprises a pentapeptide coresequence selected from the group consisting of FFFAF, YFAFF, YFYFA,YWAFF, WGYAF, WGYFA and WAFFA.
 41. A method of reducing the binding ofTGFβ1 to a TNFβ1 receptor comprising delivering to the mammal aD-peptide comprising a pentapeptide core selected from the groupconsisting of FFFXaa₁W, FWFXaa₁Xaa₂, FYXaa₁YF, FWXaa₁Xaa₂Xaa₃, FXaa₁YYW,FXaa₁YYXaa₂, FWXaa₁WY, FFWYW, FXaa₁Xaa₂FXaa₃, FYWXaa₁Y, FYWXaa₁W,FXaa₁YFXaa₂, FYYYXaa₁, FWXaa₁FF and FFXaa₁WW wherein Xaa₁, Xaa₂ and Xaa₃are amino acids of the D- or L-configuration independently selected fromthe group consisting of D, E, K, R, H, N, Q, C, S, T, G, A, V, L, I, Mand P.
 42. The method of claim 41, wherein the D-peptide comprisespentapeptide core sequence selected from the group consisting of FFFGW,FWFGA, FYGYF, FWAAA, FAYYW, FGYYG, FWAWY, FFWYW, FAAFG, FYWAY, FYWGW,FAYFG, FYYYA, FWGFF and FFAWW.
 43. The library of claim 5, wherein eachD-peptide is attached to a solid support.
 44. The library of claim 31,wherein the solid support is attached to a bead.
 45. The library ofclaim 31, wherein each peptide is attached to a microtiter plate.